We have isolated and characterized four reiteration mutants of SV40 which have amplified progressively smaller fractions of the viral genome in tandemly repeating structures. The physical mapping and biological fate of the monomeric segments from these mutant DNAs will be investigated in order to demonstrate the evolutionary (non-Darwinian) relationship among these mutants. Through the use of two restriction nucleases (endo R.EcoRI and R.Hind III) which yield cohesive termini after cleavage, we shall attempt to construct reiteration mutants both from wild-type SV40 DNA and by excision of fragments from those mutants which we have isolated. One use of the monomeric segments from these mutants will be for the propagation of foreign DNA linked to the segment in mammalian cells. These mutant DNAs have tandemly repeated the origin of viral DNA replication. They will be examined in an in vitro DNA synthesis system for possible specific initiation. We have partially purified a superhelical DNA unwinding protein from monkey kidney cells. We shall complete this purification and compare this enzyme activity from uninfected cells to that from SV40-infected cells.